Success in cloning a full-length DEN4 cDNA which can be used to produce infectious RNA transcripts has created opportunities for detailed molecular analysis of the RNA genome and for development of new dengue vaccine strategies. A panel of 3' noncoding (NC) deletion mutants was previously recovered from RNA-transfected simian LLC-MK2 cells. Most 3'NC deletion mutants exhibited restriction of growth and plaque formation in simian LLC-MK2 cells. Interestingly, mutant 3'd 303-183 produced small plaques on mosquito C6/36 cells but grew in simian LLC-MK2 cells to a high titer similar to that of wild type virus. To extend our vaccine strategy, mutant 3'd 303-183 and four other moderately restricted 3'd mutants were selected for evaluation of their infectivity and immunogenicity in rhesus monkeys. Mutant 3'd 303-183 induced a high level antibody response similar to that of the wild type virus, whereas other mutants induced low to moderate levels of antibodies as measured by radio-immunoprecipitation and virus neutralization. Unexpectedly, the results of a later preclinical trial showed that three 3' NC deletion mutants selected as candidate vaccines and the wild type DEN4 control, all prepared from cDNA and produced under GMP conditions failed to elicit an antibody response in monkeys. Experiments are in progress to determine if loss of immunogenicity of vaccine candidates could be explained by genetic alteration of DEN4 that might have occurred during recovery of dengue virus from mosquito cells transfected with RNA transcripts or during subsequent propagation of virus in simian FRhL cells that are approved for use in experimental vaccines destined for study in humans. In other studies, DEN4 cDNA was used to engineer deletions in the 5'NC region for functional analysis and for isolation of mutants that might show promise as candidate live vaccines. Several 5' NC deletion mutants that exhibited low to moderate efficiency for translation in vitro produced small plaques and exhibited reduced growth in cell culture. Among mutant constructs tested, deletion of nt 82-87 in the 5'NC region most severely reduced translation efficiency. Nevertheless, an infectious virus was recovered from simian LLC-MK2 cells transfected with the RNA transcripts of mutant 5'd 82-87. The progeny virus produced small plaques on simian LLC-MK2 cells and grew to low titer in these cells. Unlike wild type DEN4 or other 5' NC deletion mutants, mutant 5'd 82-87 failed to produce plaques on mosquito C6/36 cells and was also replication- defective in A. aegypti or A. albopictus following intrathoracic inoculation.